. Glutathione is considered a key player in protecting organisms against oxygen toxicity. However, it is absent in mycobacteria which are highly resistant to oxidative stress indicating that they use alternative thiol-protective mechanisms. It has been shown that mycobacteria produce considerable levels of a novel cysteine derivative named mycothiol which is resistant to oxidation. The investigators have shown that M. smegmatis is an order of magnitude more resistant to the thiol-oxidizing agent diamide than E. coli and other mammalian cells. This resistance appears to be due to their ability to produce mycothiol. The goal of this project is elucidate the pathways involved in mycothiol biosynthesis and utilization and to identify potential targets for TB drug development. The specific aims are designed 1) to develop a chemical synthesis of mycothiol and its components for use in testing the biosynthesis and function of mycothiol, 2) to determine the pathway of mycothiol biosynthesis, 3) to purify the coA cysteine acetyl-transferase activity detected in M. Smegmatis and determine whether it functions in the initial step of mycothiol biosynthesis, 4) to determine the spectrum of enzyme activity utilizing mycothiol substrate, and 5) to obtain mutants blocked in the synthesis of mycothiol and establish their sensitivity to oxidative challenge.